There are approximately one million persons in the U.S. infected with HIV-1, with 40,000 new cases reported each year. While most of these patients greatly benefit from highly active antiretroviral therapy (HAART), they often develop multi-drug resistance associated with viral mutations, which causes problems for long-term treatment. Knowledge of the spectrum of HIV-1 retroviral mutations is extremely important in order to effectively plan drug treatment for these patients. However sequencing is expensive, time-consuming and does not detect low-levels of drug-resistant strains. The principal objective of this proposal is to develop a highly sensitive, cost-effective and high throughput system for detecting drug-resistant strains of HIV-1 virus based on call-free protein expression and mass spectrometry. This approach is based on the fact that drug resistant inducing mutations in HIV alter the mass of the encoded proteins. During Phase I, the overall approach will be extensively evaluated using wild type(HXB2) and mutant sequences of the drug targeted viral reverse transcriptase and protease. Peptides from regions of each protein likely to contain drug-resistant mutations will be expressed in cell-free extracts from rabbit reticulocyte or E. coli using RT-PCR amplified DNA templates. In order to reduce proteolyUc degradation of cell-free expressed peptides, ia problem revealed in preliminary studies, a high fidelity reconstituted translation system based on unmodified E. coli translation factors will be tested. Several methods for rapid peptide fragment isolation from the cell-free extract will be evaluated based on earlier work by AmberGen including: i) N-terminal epitope incorporation, ii) tRNA mediated protein engineering (TRAMPE) and iii) photocleavable biotin. Mass spectrometric analysis of the purified HIV peptides will be performed using MALDI-TOF mass spectrometry both on individual peptides and multiple peptides produced by multiplexing PCR and all downstream steps. Results will be compared to other methods of HIV retrovirus genotyping including DNA sequencing. In Phase II, a high throughput MS based system will be developed for HIV-1 drug-resistance testing based on the industry standard 96- or 384-well sample format. Dr. Richard D'Aquila, an internationally recognized expert on HIV-1 drug-resistance will serve as a consultant on this project and as a source of samples. Dr. Raymond Tellier, an infectious disease expert who has used mass spectrometry to characterize quasi-species variation in Hepatitis C Virus will also serve as a consultant.